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Biomedical subjects

A J Roberts

Publications and source records attributed to A J Roberts.

At least 73 records · Page 4Linked to original sources
PubMed1994

Sensitivity of the circadian rhythm of kainic acid-induced convulsion susceptibility to manipulations of corticosterone levels and mineralocorticoid receptor binding.

Increases in corticosterone levels have been associated with enhanced susceptibility and decreases in corticosterone levels have been associated with decreased susceptibility to convulsions in mice. The proconvulsant effects of corticosterone are believed to be mediated by central mineralocorticoid receptors (MR). Both convulsion susceptibility and plasma corticosteroid levels display circadian rhythmicity. When corticosterone levels are at their lowest, hippocampal MR binding is submaximal, whereas when corticosterone levels are at their circadian peak, hippocampal MR binding is maximal. In the present experiments the relationship between circadian rhythms of susceptibility to kainic acid-induced convulsions and plasma corticosterone levels was investigated. In addition, the effects of exogenously administered corticosterone and the MR antagonist spironolactone were examined at times of different convulsion susceptibility. In general, lower plasma corticosterone levels were associated with decreased convulsion susceptibility and higher plasma corticosterone levels were associated with greater convulsion susceptibility. Corticosterone, administered when endogenous levels were low, had a proconvulsant effect. Spironolactone, administered when corticosterone levels were higher and hippocampal MR were presumably maximally occupied, had an anticonvulsant effect. These results indicate that the circadian rhythm in susceptibility to kainic acid-induced convulsions is sensitive to manipulations of corticosterone levels and MR binding. Degree of central MR occupancy may, in part, mediate convulsion susceptibility in humans as well as laboratory animals.

Animals↗
PubMed1994

In vitro production of estradiol by bovine granulosa cells: evaluation of culture condition, stage of follicular development, and location of cells within follicles.

In vitro estradiol (E2) production by bovine granulosa cells was evaluated under several culture conditions, which included the presence or absence of fetal bovine serum (FBS; 2.5 and 10%), serum substitutes (1% Nutridoma [Boehringer-Mannheim, Indianapolis, IN], 2% UltroSer G [IBF Biotechnics, Villenue-la Garenne, France]), selenium (Se; 10 ng/ml), lipoprotein (0.25% Excyte/ml), O2 concentration (5 and 20%), and two attachment factors (Pronectin F and PepTite-2000). Dulbecco's Modified Eagle's medium:Ham's F-12 medium (1:1 mixture) containing 1 microM androstenedione, 1 microgram/ml insulin, and 0.1% BSA was the basal medium evaluated. The optimum conditions determined were the basal medium in 5% O2. These conditions were then used to ascertain whether or not E2 production by granulosa cells varied with respect to location of cells within a follicle. Follicular fluid was aspirated and centrifuged to obtain granulosa cells expected to be primarily luminal and cumulus cells. Follicles were then bisected, and remaining mural granulosa cells were removed by scraping the follicle wall with a fine plastic loop. Aspirated granulosa cells secreted more (p < 0.01) E2 than scraped cells. Production of E2 during Days 0 to 2 of culture by aspirated (0.15 +/- 0.05 ng/microgram DNA) and scraped (0.02 +/- 0.01 ng/microgram DNA) granulosa cells from small follicles (< 8 mm) was less than that by aspirated (6.30 +/- 2.20 ng/micrograms DNA) and scraped cells (1.90 +/- 1.00 ng/microgram DNA) from large follicles (> or = 8 mm). During Days 2 to 4 of culture when compared to Days 0 to 2, E2 production increased for aspirated (but not scraped) granulosa cells from small follicles (0.66 +/- 0.23 ng/microgram DNA)). In contrast, E2 production decreased (p < 0.05) over time in culture for aspirated (2.10 +/- 0.50 ng/microgram DNA) and scraped (0.16 +/- 0.07 ng/microgram DNA) granulosa cells from large follicles. Thus, granulosa cells proximal to the basement membrane may be less differentiated with regard to E2 production than cells distal to the basement membrane. In addition, aspirated granulosa cells from small follicles appear to continue to differentiate toward E2-producing cells during culture, a characteristic difficult to obtain with bovine granulosa cells.

Animals↗
PubMed1994

Relationships among concentrations of steroids, insulin-like growth factor-I, and insulin-like growth factor binding proteins in ovarian follicular fluid of beef cattle.

The relationship between ovarian follicular steroidogenesis and insulin-like growth factor binding protein (IGFBP) activity was evaluated during the follicular phase of the bovine estrous cycle. In experiment 1, follicles were collected from cyclic cows (n = 11) slaughtered at 48 h after administration of prostaglandin F2 alpha (PGF; 35 mg i.m.). In experiment 2, cows were injected twice daily with saline (control) or FSH (FSH cows; total dosage = 42 mg) from Day 2 to Day 6 (estrus = Day 0) and with PGF (35 mg i.m.) on Day 7; follicles were collected from control cows (n = 20) slaughtered at 0, 24, 48, or 72 h and from FSH cows (n = 8) at 0 and 48 h after PGF. Follicular fluid was assayed for estradiol (E2), androstenedione (A4), progesterone (P4), and insulin-like growth factor-I (IGF-I) by RIA and for IGFBP activity by ligand blotting and densitometry. Intensities of the 34-kDa (IGFBP-2), 29-27-kDa, and 22-kDa IGFBP bands in follicular fluid were nondetectable or were lower (p < 0.01) in the fluid of large (> or = 8 mm) E-active (E-A; E2 > 50 ng/ml and > P4) follicles than in large E-inactive (E-I), medium (5-7 mm), or small (< 5 mm) follicles. IGFBP-3 (44-40-kDa doublet) was unaffected by follicle stage in experiment 1, but IGFBP-3 was lower (p < 0.01) in follicular fluid of E-A vs. E-I large follicles in experiment 2. Profiles of IGFBP activity were similar in follicular fluid of small, medium, and E-I large follicles. In experiment 2, E2 concentrations in large E-A follicles increased (p < 0.01) from 0 to 48 h after the PGF injection for control cows but decreased (p < 0.01) for FSH cows, whereas follicular fluid IGFBP-2 binding activity decreased from 0 to 48 h after PGF in controls and increased in FSH cows (treatment x time, p < 0.05). IGFBP-3 binding was unaffected by FSH treatment or time after administration of PGF. Profiles of IGFBP activity in homogenates of granulosa or theca cells were similar to follicular fluid profiles except for the absence of IGFBP-3 binding activity. The disappearance of binding activities for IGFBP-2 and smaller-molecular-mass IGFBPs in E-A follicles suggests a possible regulatory role for IGFBPs in follicular maturation and on aromatase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Androstenedione↗
PubMed1994

Mineralocorticoid receptors mediate the enhancing effects of corticosterone on convulsion susceptibility in mice.

With hippocampal slice electrophysiology, direct evidence that mineralocorticoid receptors (MR) mediate excitatory effects of corticosteroids on neuronal activity was found. The present experiments extended this hypothesis to a whole animal model. The effects of manipulations of MR binding on convulsions produced by pentylenetetrazol (PTZ), strychnine and kainic acid were examined. Moderate increases in plasma corticosterone levels resulted in enhanced susceptibility to several convulsion types; decreased levels attenuated the susceptibility. The proconvulsant effects of corticosterone were inhibited by the MR antagonist, spironolactone. Convulsions typically affected by these manipulations were PTZ-induced myoclonic jerk and face and forelimb clonus and kainic acid-induced convulsions. Other convulsion types, e.g., PTZ-induced running bouncing clonus and tonic hindlimb extension and strychnine-induced convulsions, were generally unaffected by these manipulations, which suggests that central MR effects are not global. The convulsions that were sensitive to MR manipulations are believed to originate in limbic structures. These results provide the first direct evidence for a role of central MRs in the modulation of central nervous system excitability in a whole animal. Furthermore, they suggest that central MR action may be important in disruptions of excitability, such as epilepsy, drug withdrawal syndromes and arousal states in healthy animals.

Animals↗
PubMed1993

Type I corticosteroid receptors modulate PTZ-induced convulsions of withdrawal seizure prone mice.

Corticosteroids have been shown to modulate convulsion expression in humans and animals. It is hypothesized that type I corticosteroid receptors mediate the excitatory effects of corticosteroids in vivo based on low-dose efficacy of corticosterone, and differential effects of mineralocorticoids vs. glucocorticoids on convulsions. In the present experiments, the effects of altering corticosterone levels, and the role of the type I receptor in mediating these effects, were examined using pentylenetetrazol (PTZ)-induced convulsions in ethanol withdrawal seizure prone (WSP) mice. It was hypothesized that stimulation of type I receptors partially mediates the expression of tonic hindlimb extensor (THE) convulsions produced by PTZ. Aminoglutethimide, a steroid synthesis inhibitor, increased latencies to PTZ-induced THE. This anticonvulsant effect was reversed by corticosterone and the type I agonist, deoxycorticosterone (DOC), but not by the type II agonist, dexamethasone. Furthermore, two type I receptor antagonists, spironolactone and RU26752, increased latencies to PTZ-induced THE, suggesting that they have anticonvulsant action. In summary, the results of these experiments suggest that type I corticosteroid receptors are important for expression of PTZ-induced convulsions.

Aminoglutethimide↗
PubMed1993

Dissociation of the effect of aminoglutethimide on corticosterone biosynthesis from ataxic and hypothermic effects in DBA and C57 mice.

Adrenalectomy is frequently used to deplete adrenocortical hormones in physiological and receptor-binding studies in animals. However, this procedure is irreversible, removes both the cortex and medulla, and produces many negative side effects such as hypotension and hypoglycemia. Aminoglutethimide is a steroid synthesis inhibitor which depletes adrenocortical hormones without these negative effects. However, aminoglutethimide itself has been shown to produce behavioral and physiological deficits. In the present experiments, dose-response relationships were determined for the effects of aminoglutethimide on corticosterone levels, motor coordination, and body temperature in C57 and DBA mice. Aminoglutethimide (5.4-54 mg/kg) inhibited the increase in plasma corticosterone concentrations normally observed in response to restraint stress. Only at higher doses (170-1,000 mg/kg) were rotarod performance and body temperature affected. The corticosterone response to restraint stress recovered fully between 12 and 24 h after aminoglutethimide. In the present study, doses of aminoglutethimide were found that temporarily inhibit stressed corticosterone release without producing motor deficits and temperature decreases. These results indicate that aminoglutethimide is a potential substitute for adrenalectomy in studies on the effects of removal of adrenocortical hormones.

Adrenalectomy↗
PubMed1992

Genetic differences in hypothalamic-pituitary-adrenal axis responsiveness to acute ethanol and acute ethanol withdrawal.

There is a growing body of evidence suggesting that corticosteroids contribute to the increased neural excitability observed during ethanol withdrawal. In the present study, this was further investigated using mouse strains which differ in ethanol withdrawal severity. DBA/2 (DBA) mice were found to display more severe acute ethanol withdrawal seizures than C57BL/6 (C57) mice. Additionally, DBA mice showed a greater stress response than C57 mice, as measured by higher plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone, to an acute dose of ethanol. Mimicking withdrawal plasma corticosterone levels by administering corticosterone to ethanol-naive mice resulted in increases in handling-induced convulsions in the range observed during withdrawal. There did not appear to be a strain difference in sensitivity to the excitatory effects of corticosterone. In summary, the greater stress response to ethanol by DBA mice may account, in part, for the more severe ethanol withdrawal syndrome of this strain.

Adrenocorticotropic Hormone↗
PubMed1992

Induction of experimental autoimmune uveoretinitis in Lewis rats with purified recombinant human retinal S-antigen fusion protein.

Full-length human retinal cDNA for S antigen (S-ag) and for the alpha subunit of transducin (alpha-Td) were subcloned into a bacterial expression plasmid vector to generate recombinant fusion proteins with glutathione-S-transferase (GST). The recombinant GST-S-ag and rGST-alpha-Td fusion proteins were purified from bacterial extracts by continuous flow preparative gel electrophoresis under denaturing conditions, and were assessed for their ability to induce experimental autoimmune uveoretinitis (EAU). Immunization of Lewis rats with single doses of 10 micrograms-100 micrograms rGST-S-ag in Freund's complete adjuvant supplemented with Bordetella pertussis readily induced clinical signs of EAU. Immunization with GST alone did not induce EAU indicating that disease activity was ascribable to the S-ag residues in the fusion protein. Although the alpha-Td shares limited sequence homology with S-ag, the rGST-alpha-Td fusion protein was also not uveitogenic in Lewis rats. The clinical severity of EAU in Lewis rats sensitized with rGST-S-ag was found to be milder than that induced with native S-ag preparations purified from human retina. However, humoral antibody responses to sensitization with the recombinant S-ag fusion protein were of a higher magnitude than with native S-ag. The availability of recombinant preparations of human S-ag protein will be of value in studying its processing and presentation to T cells derived from patients with autoimmune retinal vasculitis.

Animals↗
PubMed1992

Pathogenicity and immunogenicity of recombinant human retinal S-antigen fusion protein.

A full-length cDNA clone to human S-antigen (HS-ag) was isolated from lambda gt 10 human retinal library and expressed as a fusion protein with glutathione S-transferase (GST) in E. Coli. Uveitogenicity and immunogenicity of recombinant GST-HS-ag fusion protein and native HS-ag were compared in EAU-susceptible Lewis rats. Recombinant HS-ag was found less uveitogenic than native HS-ag. Animals inoculated with recombinant HS-ag developed EAU on day 17, three days later than those inoculated with native HS-ag, the incidence of the disease was reduced from 80% to 58% and the score of clinical severity reduced from 2.2 to 1.3 points respectively. In contrast, rGST-HS-ag was more immunogenic than native HS-ag as it elicited four times higher levels of antibodies which reacted specifically with both antigens.

Animals↗
PubMed1992

Reduction in the levels of oral malodor precursors by hydrogen peroxide: in-vitro and in-vivo assessments.

The potential of hydrogen peroxide to reduce the levels of salivary thiol precursors of oral malodor was investigated in-vitro and in-vivo. In both cases the concentration of thiol groups was determined colorimetrically by quantitative reaction with 4,4'-bis (dimethylamino) diphenyl carbinol. Addition of volumes of hydrogen peroxide solution (containing between 0.18 and 0.90 mmol) to premeasured aliquots of saliva in-vitro, resulted in reductions in salivary thiol levels of between 53% and 75% compared to controls. This positive indication prompted an in-vivo investigation. The efficacy of a fluoride-containing test toothpaste also containing 0.67% hydrogen peroxide and 5.48% sodium bicarbonate was evaluated in a crossover study using ten male and female subjects (non-smokers). All subjects used the test product and a control fluoride dentifrice, in a random order. For the duration of the study subjects used a standard silica based toothpaste containing 1500 ppm F (as sodium monofluorophosphate) exclusively for their normal oral hygiene. On each sampling morning they refrained from oral hygiene and eating and drinking on rising. At the test facility they generated a background saliva sample stimulated by chewing unflavored, unsweetened gum. Subjects brushed for 1 minute with 1.50 (+/- 0.05) g test or control paste and generated another saliva sample as before, 30 minutes after product application. Using the same analytical procedures the mean (+/- SEM) percent reduction in salivary thiol levels post treatment compared to baseline was found to be 59.0 (+/- 7.0)% for the test product compared with 12.5 (+/- 5.2)% for the fluoride control paste.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1991

Quantity and quality of embryos collected from mice passively immunized against estradiol.

The objective of this study was to determine the effects of passive immunization against estradiol on the occurrence and timing of estrus, ovulation and fertilization rates and on early embryonic development in mice. Swiss Webster female mice were randomly assigned to one of the three treatment groups to be injected with 0.1 ml saline (control; n=15), 0.1 ml anti-estradiol antisera (high dose: n=17) or 0.1 ml antiestradiol antisera diluted 1:10 with saline (low dose; n=17) at seven weeks of age. Immediately after injection mice were placed with males and observed daily for the presence of vaginal plugs for 10 d. Three days after vaginal plugs were observed, mice were terminated and the uteri were removed and flushed to determine the number and quality of unfertilized ova and embryos. No differences were observed in the timing of vaginal plug formation, the proportion of mice with vaginal plugs, or the mean number of unfertilized ova or embryos collected from each treatment group. However, the proportion of excellent or good quality embryos was reduced in the high dosage treatment. It was concluded from this study that passive immunization of mice against estradiol did not increase the number of embryos obtained from mice and that a high dosage of antisera against estradiol reduced embryo quality. These results provide evidence that alterations in levels of estradiol may adversely affect embryonic development.

Journal Article↗
PubMed1991

Reproductive and endocrine changes in ewes actively immunized against estrogens and androgens.

Twenty ewes were divided equally into two treatments to be actively immunized simultaneously against estrogen and androgen conjugated to ovalbumin (steroid immunized) or immunized against ovalbumin alone (control) to determine the effects of steroid immunization on reproduction and pituitary function. Ewes were immunized at week 0, 3 and 5 of the 93-week study and exposed to a fertile ram for two breeding seasons (weeks 7-25 and weeks 60-67). Steroid immunization reduced the proportion of ewes which exhibited estrus during the first breeding season when antibody activity against steroids was highest and reduced the proportion of ewes lambing after both breeding seasons. Ovulation rate, observed only during the second breeding season, was two times higher in steroid immunized ewes, but no increase in percent lamb crop per ewe lambing was observed. Pituitary concentrations of immunoactive LH (determined by radioimmunoassay), bioactive LH (determined by stimulation of mouse Leydig cell production of testosterone) and receptors for LHRH were increased by steroid immunization. These changes in pituitary LH resulted in a decreased ratio of bioactive to immunoactive LH observed in the pituitary and a decrease in circulating concentrations of bioactive LH. It was concluded from this study that simultaneous active immunization against estrogens and androgens increases ovulation; however, estrus and lambing rates may be reduced when antibody activity is high. Ovulation rates were increased despite the fact that no change in FSH was observed and the biopotency of LH was decreased in steroid immunized ewes.

Androgens↗
PubMed1991

Differential modulation by the stress axis of ethanol withdrawal seizure expression in WSP and WSR mice.

Withdrawal from both acute and chronic ethanol (EtOH) exposure is associated with increased neural excitability and increased activity of the hypothalamic-pituitary-adrenal axis. There is some evidence that glucocorticoids are necessary for EtOH withdrawal seizure expression. Lines of mice that were selected for severe (WSP) and minimal (WSR) EtOH withdrawal (as estimated from handling-induced convulsion scores) have been shown to differ in their stress response following an acute dose of EtOH. In this study we provide evidence that these lines of mice also differ in their sensitivity to the excitatory effects of glucocorticoids. EtOH withdrawal seizures of WSP mice were significantly increased by chronic and acute corticosterone treatment, whereas those of the WSR mice were unaffected. Neural excitability was decreased in the WSP mice when aminoglutethimide, a glucocorticoid synthesis blocker, was administered. Thus, it appears that genetic differences in EtOH withdrawal seizure severity may be due, in part, to differences in sensitivity to the excitatory effects of glucocorticoids.

Alcohol Withdrawal Delirium↗
PubMed1991

Transforming growth factor-alpha and -beta differentially regulate growth and steroidogenesis of bovine thecal cells during antral follicle development.

The actions and interactions of transforming growth factor-alpha (TGF alpha) and TGF beta on growth and differentiation of bovine thecal cells were investigated. Bovine thecal interna cells were isolated from small (less than 5 mm), medium (5-10 mm), and large (greater than 10 mm) antral follicles and cultured in the presence or absence of TGF alpha and/or TGF beta. Both [3H]thymidine incorporation and changes in cell number (i.e. DNA levels) were evaluated to determine effects on thecal cell growth. Short term treatment of cells with TGF alpha (18-24 h) stimulated thymidine incorporation, and longer term treatments (4 days) increased cell number. TGF beta suppressed thymidine incorporation below that observed in untreated cultures, but had no effect on cell number. When combined with TGF alpha, TGF beta suppressed the ability of TGF alpha to stimulate thymidine incorporation and increase cell number. The response to these growth factors was similar for cells isolated from the different stages of antral follicle development. The effects of TGF alpha and TGF beta on thecal cell differentiation were evaluated by quantitating changes in androstenedione and progesterone accumulation in cultures treated with TGFs in the absence (basal) or presence of hCG, estradiol (E2), or a combination of hCG and E2. E2 and hCG were included in this study because previous research has demonstrated that these hormones alter thecal cell steroidogenesis. Treatment with TGF alpha resulted in a suppression of basal and hormonally stimulated accumulation of androstenedione during days 0-3 of culture, whereas TGF beta did not significantly alter androstenedione accumulation. TGF alpha also suppressed progesterone accumulation during days 0-3 of culture in the absence or presence of hormones. In contrast, TGF beta stimulated accumulation of progesterone in cultures that did not contain E2, which suppressed progesterone during this period. Therefore, during days 0-3 of culture, TGF alpha appears to have suppressive effects on androstenedione and progesterone production, whereas TGF beta can stimulate progesterone production in the absence of E2. During days 3-6 of culture, thecal cell differentiation changes, and the capacity to produce androstenedione dramatically declines, while the capacity to produce progesterone increases. During this period, either TGF alpha or TGF beta slightly increased basal progesterone accumulation and partially suppressed the ability of hCG to stimulate progesterone. The effects of TGFs on thecal cell steroidogenesis were similar with cells isolated from the different stages of antral follicle development. Results from these studies provide evidence that THF alpha and TGF beta can modulate thecal cell growth and differentiation (i.e. steroidogenesis).(ABSTRACT TRUNCATED AT 400 WORDS)

Androstenedione↗
PubMed1990

Mesenchymal-epithelial cell interactions in the ovary: estrogen-induced theca cell steroidogenesis.

The role of mesenchymal-epithelial cell interactions in the control of ovarian physiology was investigated Theca cells are the mesenchymal (i.e. stromal) like cells that surround the ovarian follicle and produce androgen in response to the gonadotropin luteinizing hormone (LH). Granulosa cells are the epithelial-like cells that form the follicle, support the developing oocyte, and utilize androgens produced by theca cells as a substrate for the production of estrogen Observations presented indicate that estrogen produced by granulosa cells dramatically stimulates androgen production by theca cells Estrogen was found to have greater stimulatory effect on theca cell androgen production than gonadotropin, and a combination of estrogen and gonadotropin results in a greater than additive response of the two hormones. Regulation of androgen production by estrogen provides a local feedback loop in the follicle that will significantly influence ovarian steroidogenesis. This steroid-mediated theca-granulosa cell interaction provides evidence for the importance of mesenchymal (i.e. stromal)-epithelial cell interactions in adult tissues and implies that epithelial cells can produce paracrine factors that modulate mesenchymal cell function and differentiation. The theca cell-granulosa cell interaction identified is postulated to be a critical mesenchymal-epithelial cell interaction for the control of ovarian physiology and the endocrine status of the female.

Androgens↗
PubMed1990

Molecular cloning of a human thyrotropin receptor cDNA fragment. Use of highly degenerate, inosine containing primers derived from aligned amino acid sequences of a homologous family of glycoprotein hormone receptors.

Autoantibodies to the thyrotropin (TSH) hormone receptor (TSH-R) are present in the sera of patients with thyroid autoimmune disease which are pathogenetic leading to hyperthyroidism of Graves' disease. Considerable interest has been focused on the cloning of the human TSH-R, which has until very recently, proven exceedingly difficult due to the very low receptor level expression on thyroid cells. We have used polymerase chain reaction and highly degenerate, inosine containing oligonucleotides derived from sequence alignments of the transmembrane regions 2 and 7 of a number of G-binding protein receptors including the lutropin/choriogonadotropin (LH/CG) receptors to amplify various cDNAs from human thyroid cDNA. Sequencing analysis of 27 different clones revealed that they fall into eight different groups. The very recent publication of the complete nucleotide sequence of the human TSH-R revealed that one of the groups (GT1) containing seven clones which had been sequenced belong to the human TSH-receptor. The sequence of all 7 GT1 clones was identical and in complete concordance with transmembrane regions 2 and 7 of the published TSH-R sequence. Our results show that by designing oligonucleotides to common transmembrane regions of G-binding proteins where the primers are biased in their sequence to the LH/CG receptors it is possible to amplify the TSH-R receptor sequence.

Amino Acid Sequence↗